A Fluorogenic System for Measuring Cytosolic Localization

项目摘要 生物分子正在成为一类重要的治疗学。生物分子使令人兴奋的新疗法成为可能 包括靶向蛋白质与多肽和微小蛋白质的相互作用，使用Cas9进行基因编辑的方法 复合体，以及使用化学修饰的RNA敲除mRNA。将这些大分子输送到 胞浆是至关重要的。然而，对细胞渗透性的定量评估仍然具有挑战性。最近， Kritzer实验室报告称，CAPA(氯代烷渗透分析)可以应对这一挑战。在这项测试中，一种 氯代烷连接物连接到感兴趣的生物分子上，并与稳定表达HaloTag的 HeLa细胞系。当生物分子到达胞浆时，氯代烷连接物共价结合到HaloTag上。 然后，这些细胞与氯代烷染料孵育，该染料与任何剩余的卤代标记物发生反应。洗涤后 除去未反应的染料，然后用流式细胞术分析细胞。使用这种方法，荧光强度是 与生物分子的细胞渗透量成反比。 尽管CAPA取得了成功，但该分析最终在范围上受到了限制。它们之间的反向关系 胞浆定位和荧光强度限制分析灵敏度和必要的洗涤步骤防止 活体应用。为了克服这些缺点，我们建议开发一个荧光CAPA系统。在……里面 这种荧光染料将被连接到感兴趣的生物分子上，并开启荧光。 在胞浆中与HaloTag发生共价反应。此前报道的卤代Tag荧光染料很少。 或类似的体系，这些染料表现出低荧光开启和/或缓慢的结合动力学- 这些参数使它们不太适合基于细胞的分析。我们将第一次将分子 进化以优化HaloTag-染料对，目标是生产出一种系统，使 HaloTag-染料反应的荧光，反应动力学类似于WT HaloTag。成功完成 建议的项目将产生一种荧光卤代标签系统，该系统将极大地提高产量， CAPA方法的灵敏度和时间分辨率。从长远来看，荧光染料-HaloTag对将使 活体试验，如测量活体动物的胞液释放和药代动力学，以及一种方法 这可以直接测量时间分辨的细胞渗透率。拟议的项目将提供新的方法来 有利于药物开发管道，推进生物分子作为人类疾病的治疗手段。

Project Summary Biomolecules are emerging as an important class of therapeutics. Biomolecules enable exciting new therapies including targeting protein-protein interactions with peptides and mini-proteins, gene editing methods with Cas9 complexes, and mRNA knockdown using chemically modified RNA. Delivery of these macromolecules to the cytosol is critical. However, quantitative assessment of cell-penetration remains challenging. Recently, the Kritzer lab reported CAPA, the Chloroalkane Penetration Assay, to address this challenge. In this assay, a chloroalkane linker is attached to a biomolecule of interest and incubated with a stable HaloTag-expressing HeLa cell line. As the biomolecule reaches the cytosol, the chloroalkane linker covalently binds to HaloTag. The cells are then incubated with a chloroalkane-dye that reacts with any remaining HaloTag. After washing away unreacted dye, cells are then analyzed using flow cytometry. Using this method, fluorescence intensity is inversely proportional to the amount of cellular penetration of the biomolecule. Despite the success of CAPA, the assay is ultimately limited in scope. The inverse relationship between cytosolic localization and fluorescence intensity limits assay sensitivity and the necessary wash steps prevent in vivo applications. To circumvent these shortcomings, we propose developing a fluorogenic CAPA system. In this assay a fluorogenic dye will be conjugated to a biomolecule of interest and fluorescence will turn-on upon covalent reaction with HaloTag in the cytosol. Few fluorogenic dyes have been previously reported for HaloTag or similar systems, and these dyes display low fluorescence turn-on and/or slow conjugation kinetics – parameters that make them poorly suited for cell-based assays. For the first time, we will apply molecular evolution to optimize HaloTag-dye pairs, with the goal of producing a system with 1000-fold increase in fluorescence upon HaloTag-dye reaction, and reaction kinetics similar to WT HaloTag. Successful completion of the proposed project will yield a fluorogenic HaloTag system that will greatly improve the throughput, sensitivity, and time resolution of the CAPA method. Long-term, a fluorogenic dye-HaloTag pair will enable in vivo assays such as measuring cytosolic delivery and pharmacokinetics in live animals, as well as a methods that can measure directly time-resolved cellular penetration. The proposed project will provide new methods to benefit the drug development pipeline, advancing biomolecules as treatments for human diseases.